The Experts below are selected from a list of 8268 Experts worldwide ranked by ideXlab platform
Claudio Luchinat - One of the best experts on this subject based on the ideXlab platform.
-
magnetic susceptibility and paramagnetism based nmr
Progress in Nuclear Magnetic Resonance Spectroscopy, 2019Co-Authors: Giacomo Parigi, Enrico Ravera, Claudio LuchinatAbstract:Abstract The magnetic interactions between the nuclear magnetic moment and the magnetic moment of unpaired electron(s) depend on the structure and dynamics of the molecules where the Paramagnetic Center is located and of their partners. The long-range nature of the magnetic interactions is thus a reporter of invaluable information for structural biology studies, when other techniques often do not provide enough data for the atomic-level characterization of the system. This precious information explains the flourishing of paramagnetism-assisted NMR studies in recent years. Many Paramagnetic effects are related to the magnetic susceptibility of the Paramagnetic metal. Although these effects have been known for more than half a century, different theoretical models and new approaches have been proposed in the last decade. In this review, we have summarized the consequences for NMR spectroscopy of magnetic interactions between nuclear and electron magnetic moments, and thus of the presence of a magnetic susceptibility due to metals, and we do so using a unified notation.
-
ultrafast mas solid state nmr permits extensive 13c and 1h detection in Paramagnetic metalloproteins
Journal of the American Chemical Society, 2010Co-Authors: Ivano Bertini, Claudio Luchinat, Lyndon Emsley, Moreno Lelli, Jiafei Mao, Guido PintacudaAbstract:We show here that by combining tailored approaches based on ultrafast (60 kHz) MAS on the Co(II)-replaced catalytic domain of matrix metalloproteinase 12 (CoMMP-12) we can observe and assign, in a highly Paramagnetic protein in the solid state, (13)C and even (1)H resonances from the residues coordinating the metal Center. In addition, by exploiting the enhanced relaxation caused by the Paramagnetic Center, and the low power irradiation enabled by the fast MAS, this can be achieved in remarkably short times and at very high field (21.2 T), with only less than 1 mg of sample. Furthermore, using the known crystal structure of the compound, we are able to distinguish and measure pseudocontact (PCS) contributions to the shifts up to the coordinating ligands and to unveil structural information.
-
paramagnetism based nmr restraints provide maximum allowed probabilities for the different conformations of partially independent protein domains
Journal of the American Chemical Society, 2007Co-Authors: Ivano Bertini, Giacomo Parigi, Claudio Luchinat, Yogesh K Gupta, Massimiliano Francesco Peana, Luca Sgheri, Jing YuanAbstract:An innovative analytical/computational approach is presented to provide maximum allowed probabilities (MAPs) of conformations in protein domains not rigidly connected. The approach is applied to calmodulin and to its adduct with α-synuclein. Calmodulin is a protein constituted by two rigid domains, each of them composed by two calcium-binding EF-hand motifs, which in solution are largely free to move with respect to one another. We used the N60D mutant of calmodulin, which had been engineered to selectively bind a Paramagnetic lanthanide ion to only one of its four calcium binding sites, specifically in the second EF-hand motif of the N-terminal domain. In this way, pseudocontact shifts (pcs's) and self-orientation residual dipolar couplings (rdc's) measured on the C-terminal domain provide information on its relative mobility with respect to the domain hosting the Paramagnetic Center. Available NMR data for terbium(III) and thulium(III) calmodulin were supplemented with additional data for dysprosium(III...
-
chemical shift based constraints for solution structure determination of Paramagnetic low spin heme proteins with bis his and his cn axial ligands the cases of oxidized cytochrome b 5 and met80ala cyano cytochrome c
Journal of Biological Inorganic Chemistry, 2002Co-Authors: Lucia Banci, Ivano Bertini, Gabriele Cavallaro, Claudio LuchinatAbstract:The chemical shifts of the methyl protons of protoporphyrin IX, which are readily assigned, are related to the structural features of the axial histidine ligands in heme proteins with bis-His or His-CN axial coordination (Bertini I, Luchinat C, Parigi G, Walker FA (1999) JBIC 4:515–519). In the present paper, a module is developed which transforms the chemical shifts into a pseudo-potential energy that is a function of the dihedral angles defining the orientation of the axial ligand planes. Minimization of this pseudo-potential energy, together with the energetic contributions provided by the other constraints, yields structures consistent with the heme methyl chemical shifts. Oxidized cytochrome b 5 from rat and the cyanide derivative of the M80A mutant of yeast cytochrome c are used for test calculations. In the case of scarcity of NOEs for the axial ligands, owing to the presence of the Paramagnetic Center, the above structural constraints are shown to be quite precious. The newly refined structures are deposited in the PDB.
-
sequence specific assignment of the 1h and 15n nuclear magnetic resonance spectra of the reduced recombinant high potential iron sulfur protein i from ectothiorhodospira halophila
FEBS Journal, 1994Co-Authors: Ivano Bertini, Claudio Luchinat, Mario Piccioli, Isabella C. Felli, Dieter H W Kastrau, Maria Silvia ViezzoliAbstract:A 1H and 15N NMR investigation through two-dimensional and three-dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are Paramagnetic with a relatively low μeff of about 0.8 μB/iron ion, but the Paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closest to the Paramagnetic Center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X-ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a Paramagnetic protein to date. Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter-residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein. These data are meaningful for the detection of structural differences among mutants in future studies.
Ivano Bertini - One of the best experts on this subject based on the ideXlab platform.
-
Rapid Measurement of Pseudocontact Shifts in Metalloproteins by Proton-Detected Solid-State NMR Spectroscopy
Journal of the American Chemical Society, 2012Co-Authors: Michael J. Knight, Ivano Bertini, Lyndon Emsley, Isabella C. Felli, Roberta Pierattelli, Torsten Herrmann, Guido PintacudaAbstract:Pseudocontact shifts (PCSs) arise in para-magnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the Paramagnetic Center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of H-1-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved H-1 PCSs, in a large metalloprotein, Co2+-substituted superoxide dismutase (Co2+-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with H-1-H-1 distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.
-
ultrafast mas solid state nmr permits extensive 13c and 1h detection in Paramagnetic metalloproteins
Journal of the American Chemical Society, 2010Co-Authors: Ivano Bertini, Claudio Luchinat, Lyndon Emsley, Moreno Lelli, Jiafei Mao, Guido PintacudaAbstract:We show here that by combining tailored approaches based on ultrafast (60 kHz) MAS on the Co(II)-replaced catalytic domain of matrix metalloproteinase 12 (CoMMP-12) we can observe and assign, in a highly Paramagnetic protein in the solid state, (13)C and even (1)H resonances from the residues coordinating the metal Center. In addition, by exploiting the enhanced relaxation caused by the Paramagnetic Center, and the low power irradiation enabled by the fast MAS, this can be achieved in remarkably short times and at very high field (21.2 T), with only less than 1 mg of sample. Furthermore, using the known crystal structure of the compound, we are able to distinguish and measure pseudocontact (PCS) contributions to the shifts up to the coordinating ligands and to unveil structural information.
-
paramagnetism based nmr restraints provide maximum allowed probabilities for the different conformations of partially independent protein domains
Journal of the American Chemical Society, 2007Co-Authors: Ivano Bertini, Giacomo Parigi, Claudio Luchinat, Yogesh K Gupta, Massimiliano Francesco Peana, Luca Sgheri, Jing YuanAbstract:An innovative analytical/computational approach is presented to provide maximum allowed probabilities (MAPs) of conformations in protein domains not rigidly connected. The approach is applied to calmodulin and to its adduct with α-synuclein. Calmodulin is a protein constituted by two rigid domains, each of them composed by two calcium-binding EF-hand motifs, which in solution are largely free to move with respect to one another. We used the N60D mutant of calmodulin, which had been engineered to selectively bind a Paramagnetic lanthanide ion to only one of its four calcium binding sites, specifically in the second EF-hand motif of the N-terminal domain. In this way, pseudocontact shifts (pcs's) and self-orientation residual dipolar couplings (rdc's) measured on the C-terminal domain provide information on its relative mobility with respect to the domain hosting the Paramagnetic Center. Available NMR data for terbium(III) and thulium(III) calmodulin were supplemented with additional data for dysprosium(III...
-
chemical shift based constraints for solution structure determination of Paramagnetic low spin heme proteins with bis his and his cn axial ligands the cases of oxidized cytochrome b 5 and met80ala cyano cytochrome c
Journal of Biological Inorganic Chemistry, 2002Co-Authors: Lucia Banci, Ivano Bertini, Gabriele Cavallaro, Claudio LuchinatAbstract:The chemical shifts of the methyl protons of protoporphyrin IX, which are readily assigned, are related to the structural features of the axial histidine ligands in heme proteins with bis-His or His-CN axial coordination (Bertini I, Luchinat C, Parigi G, Walker FA (1999) JBIC 4:515–519). In the present paper, a module is developed which transforms the chemical shifts into a pseudo-potential energy that is a function of the dihedral angles defining the orientation of the axial ligand planes. Minimization of this pseudo-potential energy, together with the energetic contributions provided by the other constraints, yields structures consistent with the heme methyl chemical shifts. Oxidized cytochrome b 5 from rat and the cyanide derivative of the M80A mutant of yeast cytochrome c are used for test calculations. In the case of scarcity of NOEs for the axial ligands, owing to the presence of the Paramagnetic Center, the above structural constraints are shown to be quite precious. The newly refined structures are deposited in the PDB.
-
sequence specific assignment of the 1h and 15n nuclear magnetic resonance spectra of the reduced recombinant high potential iron sulfur protein i from ectothiorhodospira halophila
FEBS Journal, 1994Co-Authors: Ivano Bertini, Claudio Luchinat, Mario Piccioli, Isabella C. Felli, Dieter H W Kastrau, Maria Silvia ViezzoliAbstract:A 1H and 15N NMR investigation through two-dimensional and three-dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are Paramagnetic with a relatively low μeff of about 0.8 μB/iron ion, but the Paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closest to the Paramagnetic Center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X-ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a Paramagnetic protein to date. Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter-residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein. These data are meaningful for the detection of structural differences among mutants in future studies.
Susumu Takahashi - One of the best experts on this subject based on the ideXlab platform.
-
electron spin resonance spectroscopy of small ensemble Paramagnetic spins using a single nitrogen vacancy Center in diamond
Journal of Applied Physics, 2016Co-Authors: Chathuranga Abeywardana, Viktor Stepanov, Franklin H Cho, Susumu TakahashiAbstract:A nitrogen-vacancy (NV) Center in diamond is a promising sensor for nanoscale magnetic sensing. Here, we report on electron spin resonance (ESR) spectroscopy using a single NV Center in diamond. First, using a 230 GHz ESR spectrometer, we performed ensemble ESR of a type-Ib sample crystal and identified a substitutional single nitrogen impurity as a major Paramagnetic Center in the sample crystal. Then, we carried out free-induction decay and spin echo measurements of the single NV Center to study static and dynamic properties of nanoscale bath spins surrounding the NV Center. We also measured ESR spectrum of the bath spins using double electron-electron resonance spectroscopy with the single NV Center. The spectrum analysis of the NV-based ESR measurement identified that the detected spins are the nitrogen impurity spins. The experiment was also performed with several other single NV Centers in the diamond sample and demonstrated that the properties of the bath spins are unique to the NV Centers indicati...
-
electron spin resonance spectroscopy of small ensemble Paramagnetic spins using a single nitrogen vacancy Center in diamond
arXiv: Mesoscale and Nanoscale Physics, 2015Co-Authors: Chathuranga Abeywardana, Viktor Stepanov, Franklin H Cho, Susumu TakahashiAbstract:A nitrogen-vacancy (NV) Center in diamond is a promising sensor for nanoscale magnetic sensing. Here we report electron spin resonance (ESR) spectroscopy using a single NV Center in diamond. First, using a 230 GHz ESR spectrometer, we performed ensemble ESR of a type-Ib sample crystal and identified a substitutional single nitrogen impurity as a major Paramagnetic Center in the sample crystal. Then, we carried out free-induction decay and spin echo measurements of the single NV Center to study static and dynamic properties of nanoscale bath spins surrounding the NV Center. We also measured ESR spectrum of the bath spins using double electron-electron resonance spectroscopy with the single NV Center. The spectrum analysis of the NV-based ESR measurement identified that the detected spins are the nitrogen impurity spins. The experiment was also performed with several other single NV Centers in the diamond sample and demonstrated that the properties of the bath spins are unique to the NV Centers indicating the probe of spins in the microscopic volume using NV-based ESR. Finally, we discussed the number of spins detected by the NV-based ESR spectroscopy. By comparing the experimental result with simulation, we estimated the number of the detected spins to be $\leq$ 50 spins.
John L Markley - One of the best experts on this subject based on the ideXlab platform.
-
Hyperfine-Shifted 13C and 15N NMR Signals from Clostridium pasteurianum Rubredoxin: Extensive Assignments and Quantum Chemical Verification
2015Co-Authors: I-jin Lin, Timothy E Machonkin, William M Westler, Bin Xia, David S. King, John L MarkleyAbstract:Stable isotope-labeling methods, coupled with novel techniques for detecting fast-relaxing NMR signals, now permit detailed investigations of Paramagnetic Centers of metalloproteins. We have utilized these advances to carry out comprehensive assignments of the hyperfine-shifted 13C and 15N signals of the rubredoxin from Clostridium pasteurianum (CpRd) in both its oxidized and reduced states. We used residue-specific labeling (by chemical synthesis) and residue-type-selective labeling (by biosynthesis) to assign signals detected by one-dimensional 15N NMR spectroscopy, to nitrogen atoms near the iron Center. We refined and extended these 15N assignments to the adjacent carbonyl carbons by means of one-dimensional 13C[15N] decoupling difference experiments. We collected Paramagnetic-optimized SuperWEFT 13C[13C] constant time COSY (SW-CT-COSY) data to complete the assignment of 13C signals of reduced CpRd. By following these 13C signals as the protein was gradually oxidized, we transferred these assignments to carbons in the oxidized state. We have compared these assignments with hyperfine chemical shifts calculated from available X-ray structures of CpRd in its oxidized and reduced forms. The results allow the evaluation of the X-ray structural models as representative of the solution structure of the protein, and they provide a framework for future investigation of the active site of this protein. The methods developed here should be applicable to other proteins that contain a Paramagnetic Center with high spin and slow electron exchange
-
zinc substituted desulfovibrio gigas desulforedoxins resolving subunit degeneracy with nonsymmetric pseudocontact shifts
Protein Science, 2009Co-Authors: Brian J Goodfellow, Frank Rusnak, Isabel Moura, José J. G. Moura, Sofia Nunes, Carla Ascenso, Brian F Volkman, John L MarkleyAbstract:Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] Center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal Center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a Paramagnetic Center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.
-
strategy for the study of Paramagnetic proteins with slow electronic relaxation rates by nmr spectroscopy application to oxidized human 2fe 2s ferredoxin
Journal of the American Chemical Society, 2004Co-Authors: Timothy E Machonkin, William M Westler, John L MarkleyAbstract:NMR studies of Paramagnetic proteins are hampered by the rapid relaxation of nuclei near the Paramagnetic Center, which prevents the application of conventional methods to investigations of the most interesting regions of such molecules. This problem is particularly acute in systems with slow electronic relaxation rates. We present a strategy that can be used with a protein with slow electronic relaxation to identify and assign resonances from nuclei near the Paramagnetic Center. Oxidized human [2Fe-2S] ferredoxin (adrenodoxin) was used to test the approach. The strategy involves six steps: (1) NMR signals from (1)H, (13)C, and (15)N nuclei unaffected or minimally affected by Paramagnetic effects are assigned by standard multinuclear two- and three-dimensional (2D and 3D) spectroscopic methods with protein samples labeled uniformly with (13)C and (15)N. (2) The very broad, hyperfine-shifted signals from carbons in the residues that ligate the metal Center are classified by amino acid and atom type by selective (13)C labeling and one-dimensional (1D) (13)C NMR spectroscopy. (3) Spin systems involving carbons near the Paramagnetic Center that are broadened but not hyperfine-shifted are elucidated by (13)C[(13)C] constant time correlation spectroscopy (CT-COSY). (4) Signals from amide nitrogens affected by the Paramagnetic Center are assigned to amino acid type by selective (15)N labeling and 1D (15)N NMR spectroscopy. (5) Sequence-specific assignments of these carbon and nitrogen signals are determined by 1D (13)C[(15)N] difference decoupling experiments. (6) Signals from (1)H nuclei in these spin systems are assigned by Paramagnetic-optimized 2D and 3D (1)H[(13)C] experiments. For oxidized human ferredoxin, this strategy led to assignments (to amino acid and atom type) for 88% of the carbons in the [2Fe-2S] cluster-binding loops (residues 43-58 and 89-94). These included complete carbon spin-system assignments for eight of the 22 residues and partial assignments for each of the others. Sequence-specific assignments were determined for the backbone (15)N signals from nine of the 22 residues and ambiguous assignments for five of the others.
-
13c 13c 2d nmr a novel strategy for the study of Paramagnetic proteins with slow electronic relaxation rates
Journal of the American Chemical Society, 2002Co-Authors: Timothy E Machonkin, William M Westler, John L MarkleyAbstract:Oxidized human [2Fe-2S] ferredoxin has a notably slow electronic relaxation rate, which precludes the observation of signals from nuclei near the iron−sulfur cluster by conventional 2D or 3D methods that utilize proton detection. We have demonstrated the utility of 13C{13C} CT-COSY in identifying connectivity information from fast relaxing carbon nuclei near the Paramagnetic Center, including those from residues that ligate the metal Center.
Giacomo Parigi - One of the best experts on this subject based on the ideXlab platform.
-
magnetic susceptibility and paramagnetism based nmr
Progress in Nuclear Magnetic Resonance Spectroscopy, 2019Co-Authors: Giacomo Parigi, Enrico Ravera, Claudio LuchinatAbstract:Abstract The magnetic interactions between the nuclear magnetic moment and the magnetic moment of unpaired electron(s) depend on the structure and dynamics of the molecules where the Paramagnetic Center is located and of their partners. The long-range nature of the magnetic interactions is thus a reporter of invaluable information for structural biology studies, when other techniques often do not provide enough data for the atomic-level characterization of the system. This precious information explains the flourishing of paramagnetism-assisted NMR studies in recent years. Many Paramagnetic effects are related to the magnetic susceptibility of the Paramagnetic metal. Although these effects have been known for more than half a century, different theoretical models and new approaches have been proposed in the last decade. In this review, we have summarized the consequences for NMR spectroscopy of magnetic interactions between nuclear and electron magnetic moments, and thus of the presence of a magnetic susceptibility due to metals, and we do so using a unified notation.
-
how do nuclei couple to the magnetic moment of a Paramagnetic Center a new theory at the gauntlet of the experiments
Journal of Physical Chemistry Letters, 2019Co-Authors: Linda Cerofolini, Enrico Ravera, Jose Silva, Maurizio Romanelli, Carlos F G C Geraldes, Anjos L Macedo, Marco Fragai, Giacomo ParigiAbstract:The recent derivation, based on pure quantum chemistry (QC) first-principles, of the pseudocontact shifts (PCSs) caused by a Paramagnetic metal Center on far away nuclei has cast doubts on the validity of the semiempirical (SE) theory, predicting PCSs to arise from the metal magnetic susceptibility anisotropy. The SE theory has been used and applied countless times, especially in the last 2 decades, to obtain structural information on proteins containing Paramagnetic metal ions. We show here that the QC and SE predictions can be directly tested against experiments, provided a suitable macromolecular system is used. The SE approach yields a good prediction of the experimental PCSs while the QC one does not. It appears that the classic theory is able to grasp satisfactorily the underlying physics.
-
paramagnetism based nmr restraints provide maximum allowed probabilities for the different conformations of partially independent protein domains
Journal of the American Chemical Society, 2007Co-Authors: Ivano Bertini, Giacomo Parigi, Claudio Luchinat, Yogesh K Gupta, Massimiliano Francesco Peana, Luca Sgheri, Jing YuanAbstract:An innovative analytical/computational approach is presented to provide maximum allowed probabilities (MAPs) of conformations in protein domains not rigidly connected. The approach is applied to calmodulin and to its adduct with α-synuclein. Calmodulin is a protein constituted by two rigid domains, each of them composed by two calcium-binding EF-hand motifs, which in solution are largely free to move with respect to one another. We used the N60D mutant of calmodulin, which had been engineered to selectively bind a Paramagnetic lanthanide ion to only one of its four calcium binding sites, specifically in the second EF-hand motif of the N-terminal domain. In this way, pseudocontact shifts (pcs's) and self-orientation residual dipolar couplings (rdc's) measured on the C-terminal domain provide information on its relative mobility with respect to the domain hosting the Paramagnetic Center. Available NMR data for terbium(III) and thulium(III) calmodulin were supplemented with additional data for dysprosium(III...
